Biomedical research is a large component of the global economy. The emphasis on molecular intervention in disease using pharmaceutical agents is a cornerstone of modern medicine. Surprisingly, the drug discovery strategy remains much the way it has been for decades, though the speed with which new compounds are examined has dramatically accelerated. The impact of “rational” drug design is only now just emerging. Knowledge of the molecular structure of potential therapeutic targets—most often proteins—is critical to the emerging rational drug design strategy and is a central component of leading-edge pharmaceutical research.
New drugs typically are developed by starting with a large pool of compounds and performing random screens against a potential target. From a pool of 10,000 compounds only about five will make it to clinical trials. This process is inefficient and costly; usually about one third of the total development cost is spent here. It has been known that structural information about the targets of these drugs can greatly enhance the discovery and development process, thus lowering the costs and development time for new medicines. Hence there is justification for funding national programs such as the Protein Structure Initiative to get this information into the public domain and make it available to all researchers.
The determination of protein structures is a vital component of our understanding of nature. In the area of medicine and drug design the structure of a protein is necessary for developing effective pharmaceuticals. There are two techniques that dominate the structure determination field. One is x-ray crystallography and the other is nuclear magnetic resonance (NMR) spectroscopy. There are of course advantages and disadvantages to both. X-ray crystallography is the oldest of the techniques and therefore is the best established in terms of methods for collecting and analyzing data. A structure determined by x-ray crystallography is a largely static picture of the protein molecule. This method has few limitations on the protein provided it can be crystallized. Therein lies one major disadvantage of this technique. It turns out that the biggest bottleneck in this method is the generation of crystals suitable for analysis. The vast majority of the potential proteins targeted for drug development are not currently amenable to this technique due to the present lack of a method for making the crystal sample.
An alternate approach to obtaining high resolution structures of proteins is nuclear magnetic resonance (NMR). NMR employs the same basic physical principles that underlies the commonly recognized medical imaging technique known as Magnetic Resonance Imaging or MRI. NMR is a powerful technique that can provide the structure of a protein in an aqueous, thus more natural, environment as opposed to a crystal. Proteins are not static entities; rather they are dynamic. It is becoming increasingly clear that the dynamics of the protein will provide important information for refining drug design and our understanding of proteins in general. Protein sample preparation is typically less of a problem for NMR, although special reagents are necessary which add to the cost of the technique. One major disadvantage of NMR with respect to x-ray crystallography is the cost of the instrumentation and the requirement for highly skilled labor to operate the NMR spectrometer and analyze the results. Another disadvantage is that current NMR techniques can only be easily applied to proteins of limited size and many proteins of interest are outside of this range. However, new tools are reducing this limitation and NMR is experiencing a resurgence as a structure determination tool.
Solution NMR spectroscopy continues to play a central role in the characterization of the structure and dynamics of proteins, nucleic acids and their complexes. Over the past fifteen years there have been remarkable developments in NMR techniques and supporting technologies such that the comprehensive structural characterization of proteins of moderate size (about 30 kDa) has become routine (for concise reviews, see Clore, G. M., and Gronenborn, A. M. 1997. Nat. Struct. Biol. 4 Suppl: 849-853; Wagner, G., 1997, Nat. Struct. Biol. 4 Suppl: 841-844). The size of protein structures that can be solved by modern NMR techniques has dramatically increased over the past decade. Coupled with the introduction of heteronuclear (Sorensen, O. W., Eich, G. W., Levitt, M. H., Bodenhausen, G., and Ernst, R. R. 1987. Prog. NMR Spectr. 16: 163-192) and ultimately triple resonance spectroscopy (Kay, L. E., Clore, G. M., Bax, A., and Gronenborn, A. M. 1990. Science 249: 411-414), was the widespread use of recombinant technologies in order to introduce NMR-active isotopes into proteins and nucleic acids (McIntosh, L. P., and Dahlquist, F. W. 1990. Q. Rev. Biophys. 23: 1-38; LeMaster, D. M. 1994, Prog. NMR Spectr. 26: 371-419). With the development of multinuclear and multidimensional capabilities, NMR is now able to efficiently and comprehensively deal with proteins of significant size and spectral complexity and has, with the advent of Transverse Relaxation Optimized Spectroscopy, or TROSY (Pervushin, K., Riek, R., Wider, G., and Wuthrich, K. 1997. Proc. Nat. Acad. Sci. USA 94: 12366-12371), reached significant heights (Riek, R., Pervushin, K., and Wuthrich, K. 2000. Trends Biochem. Sci. 25: 462-468; Tugarinov, V., Muhandiram, R., Ayed, A., and Kay, L. E. 2002. J. Am. Chem. Soc. 124: 10025-10035).
However, increasing protein size brings with it several important limitations that unfortunately compound each other to often restrict the size of a protein that can be efficiently approached by modern NMR techniques. First, increasing size leads to slower tumbling and correspondingly shorter spin-spin relaxation times. Thereby the fundamental techniques of protein NMR spectroscopy, triple resonance and total correlation technologies, begin to fail. As lines broaden, basic sensitivity also begins to become a limiting issue. Second, increasing size leads to increasingly complex spectra. Spectral degeneracy complicates the assignment process and renders assignment of Nuclear Overhauser Effects (NOEs) and other structural restraints to parent nuclei problematic.
Briefly stated, increasing size leads to slower tumbling of the macromolecule which in turn results in more efficient dipolar relaxation processes and shorter spin-spin relaxation times. The coherence transfer processes underlying current triple resonance-based assignment strategies are time-dependent and begin to fail with proteins that are about 30 kiloDaltons (kD) and larger. Chemical approaches such as random partial- or perdeuteration have been used successfully to reduce the dipolar field so that high resolution 15N Heteronuclear Single Quantum Coherence, or 15N-HSQC, spectra can be obtained (LeMaster, D. M. 1994. Prog. NMR Spectr. 26: 371-419.). Unfortunately, perdeuteration also drastically limits the structural information available from the NOE. Fractional deuteration has limited sensitivity, and its applicability as a general solution to the dipolar broadening displayed by proteins above 35 kDa is uncertain. Spectroscopic solutions to these problems are also appearing. Some find their roots in the steady improvement in the use of the rotating frame to provide for more efficient isotropic mixing for coherence transfer. These and other recent advances such as TROSY are extremely powerful, but they do not solve all of the issues facing the solution NMR spectroscopist. The difficulty of dealing comprehensively with large proteins in a general manner remains as a significant limitation to applying solution NMR methods to the rapidly growing list of proteins being discovered by the molecular biology community.
In order to improve tumbling and spin-spin relaxation times, a solvent having a lower viscosity than that of water may be used. The basic idea is to take advantage of the linear dependence for molecular reorientation on the bulk solvent viscosity as related by the classic Stokes-Einstein relationship for a sphere:
      τ    m    =            η      ⁢                          ⁢      V        kT  
where τm is the molecular reorientation correlation time, η is the bulk solvent viscosity, V is the volume of the sphere, k is the Boltzmann constant and T is the absolute temperature. Though more complex treatments are available, the Stokes-Einstein relationship serves to illustrate the approach. By encapsulating a protein within the protective shell of a reverse micelle one can solubilize the resulting particle in a very low viscosity solvent, for example, liquid pentane, butane, propane or ethane.
Such solvents are incompatible with proteins. However, by encapsulating a protein in reverse micelles, it can be safely dissolved in such solvents without denaturation. Wand and coworkers have developed and demonstrated the basic approach using the small, stable, water-soluble protein ubiquitin (Wand, A. J., Ehrhardt, M. R., and Flynn, P. F. 1998. Proc. Nat. Acad. Sci. USA 95: 15299-15302), the structure of which was determined to very high resolution inside the reverse micelle, and was shown to match that found in aqueous solution (Babu, C. R., Flynn, P. F., and Wand, A. J. 2001. J. Am. Chem. Soc. 123: 2691-2692). This technique has since been expanded to include a variety of other soluble proteins (Lefebvre, B. G., Liu, W., Peterson, R. W., Lefebvre, B. G., and Wand, A. J. 2005, J. Magn. Reson., 175: 158-162; Peterson, R. W., Lefebvre, B. G., and Wand, A. J. 2005, J. Am. Chem. Soc. 127: 10176-10177), using surfactants other than AOT (Lefebvre, B. G., Liu, W., Peterson, R. W., Valentine, K. G., and Wand, A. J. 2005. J. Magn. Reson. 175: 158-162; Peterson, R. W., Pometun, M. S., Shi, Z., and Wand, A. J. 2005. Protein Sci. 14: 2919-2921; Shi, Z., Peterson, R. W., and Wand, A. J. 2005. Langmuir 21: 10632-10637). In addition it has been shown that proteins inside a reverse micelle in liquid ethane do in fact tumble faster than they do in water, even for small proteins such as ubiquitin (Peterson, R. W., Lefebvre, B. G., and Wand, A. J. 2005, J. Am. Chem. Soc. 127: 10176-10177).
In addition to structure determination it has become apparent that a persistent problem in drug discovery is just trying to maintain stable constructs of proteins for biochemical assays. Certain proteins such as membrane proteins, that is, proteins that associate with cell membranes either by embedding in or anchoring to the membrane, and also metastable proteins are exceptionally difficult to handle. These proteins show a distinct tendency to aggregate in solution either as free protein or as a result of ligand binding before studies can be performed. This is a major problem in structure determination. Integral membrane proteins such as G-protein coupled receptors (GPCR) are a major target of current drug therapies, accounting for approximately 50% of the current total pharmaceutical market. The structure of the first therapeutic GPCR, the β2-adrenergic GPCR, was solved only in November of 2007 (Cherezov, V., Rosenbaum Daniel, M., Hanson Michael, A., Rasmussen Soren, G. F., Thian Foon, S., Kobilka Tong, S., Choi, H.-J., Kuhn, P., Weis William, I., Kobilka Brian, K., et al. 2007. Science (New York, N.Y.) 318: 1258-1265). Obviously new methods for handling such proteins are required.
Reverse micelles have a growing track record of being uniquely suited for solubilizing proteins of all classes, and provide a robust construct by which such proteins can be studied using a variety of spectroscopic techniques. However, one of the more pressing technical challenges is being able to achieve sufficient concentrations of the protein in the reverse micelle sample to do meaningful spectroscopy.
Reverse micelles will spontaneously form as transparent solutions in a low polarity liquid, and are thermodynamically stable assemblies of surfactant molecules organized around a water core. Reverse micelles were the subject of extensive attention in the 1980s as potential devices for a range of applications including separations, chromatography and reaction processes (Golden, K. E., and Hatton, T. A. 1985. Biotechnol Progr 1: 69-74; Golden, K. E., and Hatton, T. A. 1985. Abstr Pap Am Chem S 190: 128-Mbd). More recently, they have become the focus of further attention in the context of hosting various chemical reactions in solvents with low environmental impact such as supercritical carbon dioxide (Johnston, K. P., Randolph, T., Bright, F., and Howdle, S. 1996. Science 272: 1726).
The size and stability of a reverse micelle is dependent upon the amount of water loading, the molar ratio of water to surfactant, Wo. The commonly used surfactant sodium bis(2-ethylhexyl)sulfosuccinate (AOT) has been extensively studied in this respect for a variety of organic solvents. The chemical structure of AOT is shown in FIG. 1. Water loadings have been described that yield stable reverse micelles of AOT in a variety of long and short chain alkane solvents large enough to accommodate proteins (e.g., Frank, S. G., and Zografi, G. 1969. J. Coll. Interf. Sci. 29: 27-35; Gale, R. W., Fulton, J. L., and Smith, R. D. 1987. J. Am. Chem. Soc. 109: 920-921; Fulton, J. L., and Smith, R. D. 1988. Acs Sym Ser 373: 91-107; Fulton, J. L., Blitz, J. P., Tingey, J. M., and Smith, R. D. 1989. J Phys Chem-Us 93: 4198-4204). Indeed, there have been reports that all of the three shortest alkane solvents will support formation of a single phase containing reverse micelles. Ethane requires significantly elevated pressures (up to 650 bar, see Fulton, J. L., and Smith, R. D. 1988. Acs Sym Ser 373: 91-107; Fulton, J. L., Blitz, J. P., Tingey, J. M., and Smith, R. D. 1989. J Phys Chem-Us 93: 4198-4204; Smith, R. D., Fulton, J. L., Blitz, J. P., and Tingey, J. M. 1990. J Phys Chem-Us 94: 781-787) to obtain optimal behavior while butane requires the lowest pressure (a few bar).
Reverse micelles are typically prepared in one of two ways. Since water content is directly correlated to the size of the reverse micelle, and thus the performance in NMR spectroscopy experiments, the goal has been to minimize the amount of water present. To that end, the preferred method for preparing reverse micelles is to use a concentrated stock of the protein of interest, and inject that into a reverse micelle solution whereupon the protein is spontaneously encapsulated. For the purposes of sample preparation for NMR spectroscopy, this method requires the starting protein stock to be much greater than 1 mM in concentration. Concentration of the protein can be achieved by filtration methods in the aqueous phase or by other methods such as lyophilization and resuspension of the protein in a small aliquot of water or buffer. The ability to concentrate a protein in the aqueous phase to this degree is almost certainly not feasible for complex constructs such as membrane proteins, and in reality is more of an exception than the rule for most water soluble proteins as well. Lyophilizing integral membrane constructs has also been found to be disruptive to the construct in many cases. Thus it would appear that the applicability of either of the above concentration approaches is limited to special cases.
The alternative is to use a bulk transfer method whereby an aqueous phase containing the protein is brought into contact with an equal volume of organic phase containing empty reverse micelles. Upon mild agitation the protein spontaneously transfers into the organic phase in the form of encapsulated proteins; that is, reverse micelles. The water loading (Wo) tends to be much higher using this method; however, proper selection of buffer components makes the water content a manageable problem. Unfortunately, attempts to use this method with integral membrane proteins have proven difficult. The reason for this is not known, but it is thought that the driving force for encapsulation exclusively in the organic phase is overcome by the tendency for the protein to remain at the natural interface region between the aqueous and organic phases. It has become apparent that this method for encapsulating proteins in reverse micelles generally fails for integral membrane proteins.